GSM6886592: 7031_invitro_d10_sgRNA; Homo sapiens; OTHER
For scRNA-seq, primary cells were thawed in PBS+5% FBS and treated with DNase I (AMPD1-1KT, Sigma) for 5 min at room temperature. Cells were washed and resuspended with PBS+0.04% BSA. Viability was checked by Trypan Blue (Thermo), and if viability was lower than 95%, live cells were purified by the Dead cell removal kit (MACS). Viable cells were subject to scRNA-seq following the Chromium Next GEM Single Cell 5' v2 protocol. For in vitro Perturb-seq, ells were transduced as described above and maintained in a dish for 9-14 days. For in vivo Perturb-seq, leukemia cells were harvested from BM on day 12 post-transplant. Sorted GFP/mCherry+ leukemia cells were washed 3 times and resuspended in PBS+0.04% BSA and checked for viability. Viable cells were subject to scRNA-seq following the Chromium Next GEM Single Cell 5' v2 protocol with spike-in of sgRNA amplification oligos. For scRNA-seq, samples were prepared following the Chromium Next GEM Single Cell 5' v2 protocol. For Perturb-seq, samples were subject to the Perturb-seq protocol adapted from ECCITE-seq with small modifications, without antibody-hashtag labelling. Briefly, 2μM sg_RT_v4 (AGCAAGTGAGAAGCATCGTGTCAAAGCACCGACTCGGTGCCAC) oligo was spiked in to the cell suspension containing 20,000 live cells. Cells were barcoded on the 10xGenomics platform following the Chromium Next GEM Single Cell 5' v2 protocol. sgRNA was amplified together with cDNA using a spike-in Guide-tag_additive (AGCAAGTGAGAAGCATCGTGTC), and separated from cDNA by 0.6x SPRIselect beads (Beckman) (beads bind cDNA while supernatant contains sgRNAs). sgRNA was further amplified with primers incorporated with TotalSeq_A_Dual_Index, and purified by 1.2x SPRIselect beads (Beckman). cDNA was amplified and purified following the 10x Genomics protocol. Libraries were analyzed using the Qubit dsDNA HS assay kit (Thermo Fisher) and a High Sensitivity DNA Bioanalyzer Kit (Agilent), and quantified by NEBNext Library Quant Kit for Illumina. Libraries were pooled consisting of 90% cDNA and 10% sgRNA, and pair-end sequenced on a Nova-seq 6000 in Read 1 26, i7 index 10, i5 index 10, Read 2 90bp format.